The present invention relates to a modified amino acid sequence of Protein Kinase N, and more particularly to a modified amino acid sequence of PKN having activated Rho protein binding activity.
A group of low-molecular-weight GTP-binding proteins (G-proteins) with molecular weights of 20,000-30,000 with no subunit structures are observed in organisms. To date, over fifty or more members have been found as the super family of the low-molecular-weight G-proteins in a variety of organisms, from yeast to mammals. The low-molecular-weight G-proteins are divided into four families of Ras, Rho, Rab and the others based on homologies of amino acid sequences. It has been revealed that the small G-proteins control a variety of cellular functions. For example, the Ras protein is considered to control cell proliferation and differentiation, and the Rho protein is considered to control cell morphological change, adhesion and motility.
The Rho protein, having GDP/GTP-binding activity and intrinsic GTPase activity, is believed to be involved in cytoskeletal rearrangement in response to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. When the inactive GDP-binding Rho is stimulated, it is converted to the active GTP-binding Rho protein (hereinafter referred to as xe2x80x9cthe activated Rho proteinxe2x80x9d) by GDP/GTP exchange proteins such as Smg GDS, Dbl or Ost. The activated Rho protein then acts on target proteins to form stress fibers and focal contacts, thus inducing the cell adhesion and motility (Experimental Medicine, Vol. 12, No. 8, 97-102 (1994); Takai, Y. et al., Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the intrinsic GTPase activity of the Rho protein converts the activated Rho protein to the GDP-binding Rho protein. This intrinsic GTPase activity is activated by what is called GTPase-activating proteins (GAP) (Lamarche, N. and Hall, A. et al., TIG, 10, 436-440 (1994)).
The Rho family proteins, including RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42, share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley and A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley and A. Hall, EMBO J., 13, 2600-2610 (1994)). The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995)*; Landanna, C. et al., Science, 271, 981-983 (1996)*, cell motility (K. Takaishi et al., Oncogene, 9, 273-279 (1994), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)). (An asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.) In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)*; K. Kimura, et al., Science, 273, 245-248(1996)*), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).
Recently, it has also be found that Ras-dependent tumorigenesis is suppressed when the Rho protein of which the amino acid sequence has been partly substituted is introduced to cells, revealing that the Rho protein plays an important role in Ras-induced transformation, that is, tumorigenesis (G. C. Prendergast et al., Oncogene, 10, 2289-2296 (1995); Khosravi-Far, R. et al., Mol. Cell. Biol., 15, 6443-6453 (1995)*; R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995)*; Lebowitz, P. et al., Mol. Cell, Biol., 15, 6613-6622 (1995)*).
It has also been demonstrated that mutation of GDP/GTP-exchange proteins which act on the Rho protein results in cell transformation (Collard, J., Int. J. Oncol., 8, 131-138 (1996)*; Hart, M. et al., J. Biol. Chem., 269, 62-65 (1994); Horii, Y. et al., EMBO J., 13, 4776-4786 (1994)).
In addition, the Rho protein has been elucidated to be involved in cancer cell invasion, that is, metastasis (Yoshioka, K. et al., FEBS Lett., 372, 25-28 (1995)). The cancer cell invasion is closely associated with changes in cancer cell activity to form cell adhesion. In this context, the Rho protein is also known to be involved in the formation of cell adhesion (see above Morii, N. et al. (1992); Tominaga, T. et al. (1993); Nusrat, A. et al. (1995); Landanna C. et al. (1996)*).
On the other hand, a novel protein kinase having a molecular weight of approximately 120 kDa (hereinafter referred to as PKN or Protein Kinase N) has recently been isolated, and the whole amino acid sequence thereof has been determined. Furthermore, PKN has been proved to have a catalytic region highly homologous to that of Protein Kinase C and actually has serine/threonine kinase activity (Mukai, H. and Ono, Y. Biochem. Biophys. Res. Commun. 199, 897-904 (1994), Mukai, H. et al., Biochem. Biophys. Res. Commun. 204, 348-356 (1994), and Mukai, H. et al., Biochem. Biophys. Acta 1261, 296-300 (1995)). Substitution of Arg for Lys at position 644 of PKN leads to loss of the protein kinase activity (Mukai, H. et al., ibid.).
The protein kinase activity is activated by unsaturated fatty acids such as arachidonic acid (Mukai, H. et al., Biochem. Biophys. Res. Commun., 204, 348-356 (1994); and Kitagawa, M. et al., Biochem. J., 310, 657-664 (1994)). cDNAs of PKN of human beings, rat, and Xenopus have been cloned, and the amino acid sequences thereof have been determined (Mukai, H. and Ono, Y., Biochem. Biophys. Res. Commun., 199, 897-904 (1994); Mukai, H. et al., Biochim. Biophys. Acta., 1261, 296-300 (1995)). PKN from human is a protein of 942 amino acid residues, and the amino acid sequence of the carboxyl-terminal catalytic region is highly homologous to the amino acid sequence of the catalytic region of Protein Kinase C. Therefore, PKN is often called Protein Kinase C-related kinase 1 (Palmer, R. H. and Parker, P. J., FEBS Lett., 356, 5-8 (1994)).
The amino-terminal regulatory region of PKN contains some of leucine zipper sequences, and a polybasic region is located immediately at the amino terminal side of the leucine zipper sequence. Furthermore, it has been reported that, there are at least two isozymes concerning PKN (protein kinase C-associated kinases 2 and 3) (Palmer, R. H. and Parker, P. J., FEBS Lett. 356, 5-8 (1994)).
It is only recently (after the first filed application which provides the right of the priority of the present application) that a several proteins have been identified as candidates of Rho-targets in mammals different from PKN: citron (Madaule, P. et al., FEBS Lett., 377, 243-248 (1995)*), rhophilin (Watanabe, G. et al., Science, 271, 645-648 (1996)*), p160ROCK (Ishizaki, T. et al., EMBO J. 15, 1885-1893(1996)*), Rho-associated kinase (Matsui, T. et al., EMBO J., 15, 1885-1893 (1996)*), ROKxcex1(Leung, T. et al., J. Biol. Chem., 270, 29051-29054 (1995)*), rhotekin (Reid, T. et al., J. Biol. Chem., 271, 9816-9822 (1996)*), and myosin binding subunit(K. Kimura, et al., Science 273, 245-248(1996)*). In addtion, Protein Kinase C1 (PKC1) (Nonaka, H. et al., EMBO J. 14, 5931-5938(1995)*) and 1,3-xcex2-glucan synthase (Drgonova, J. et al., Science 272, 277-279(1996)*; Qadota, H. et al., Science 272, 279-281(1996*) have been identified as candidates of Rho-targets in yeasts (Saccharromyces cerevisiae).
Microtubules, actin filaments, and intermediate filaments may be mentioned as major fibrous components constituting cytoskeleton. It is known that the cytoskeleton is controlled by the phosphorylation of these fibrous components (N. Inagaki et al., Trend. Biochem. Sci., 19, 448-452 (1994)). Furthermore, regarding the intermediate filament, the structure has been elucidated on amino acid sequence level (Julien, J. et al., Biochim. Biophys. Acta 909, 10-20 (1987) (human neurofilament-L), Myers, M. et al., EMBO J., 6, 1617-1626 (1987) (human neurofilament-M), Lees, J., et al., EMBO J., 7, 1947-1955 (1988) (human neurofilament-H), and Honore, B., et al., Nucl. Acid. Res., 18, 6692 (1990) (human vimentin)). However, insofar as the present inventors know, the interaction between the intermediate filament and PKN has not been reported.
Furthermore, a wide variety of isoforms of cytoskeletal protein xcex1-actinin, including skeletal muscle-, smooth muscle-, and non-muscle-types of xcex1-actinins derived from various cells and tissues, has been characterized. In human, only clones of skeletal muscle-type of xcex1-actinin (HuActSkl, designated in Beggs, A., et al., J. Biol. Chem. 267, 9281-9288 (1992)) and non-muscle-type of xcex1-actinin (HuActNm, designated in Beggs, A., et al., J. Biol. Chem. 267, 9281-9288 (1992)) highly homologous to HuActSkl (89% similarity and 80% identity) have been isolated (Millake, D. B., et al., Nucleic Acids Res. 17, 6725 (1989); and Youssoufian, H., et al., Am. J. Hum. Genet. 47, 62-71 (1990)). The functional difference among these xcex1-actinins is that binding of the muscle isoform to F-actin is inhibited by Ca2+, whereas binding of the non-muscle isoform is insensitive to Ca2+ (Burridge, K. and Feramiscoo, J. R. Nature 294, 565-567 (1981); Bennett, J. P., et al., Biochemistry 23, 5081-5086 (1984); Duhaiman, A. S. and Bamburg, J. R. Biochemistry 23, 1600-1608 (1984); and Landon, F., et al., Eur. J. Biochem. 153, 231-237 (1985)).
xcex1-Actinin is a member of spectrin superfamily, including spectrin and dystrophin (Blanchard, A., et al., J. Muscle Res. Cell Motil. 10, 280-289 (1989); Dubreuil, R. R. Bioessays 13, 219-226 (1991); and Bennett, V, Physiol. Rev. 70, 1029-1065 (1990)). Family members are characterized by the N-terminal actin-binding domain, central rod-shaped spectrin-like repeats, and the C-terminal EF-hand-like domain. xcex1-Spectrin contains 21 rod-shaped repeats in the N-terminal instead of the EF-hand-like domain. The C-terminus of xcex1-spectrin is clearly identical to xcex1-actinin, and especially the repeat 20 of xcex1-spectrin is highly homologous to the repeat 3 of xcex1-actinin (Wasenius, V. M., et al., J. Cell Biol. 108, 79-93 (1989); and Hong, W. J. and Doyle, D. J. Biol. Chem. 264, 12758-12764 (1989)). The positions of the repeat in these proteins are substantially identical to each other.
xcex1-Actinin is composed of three domains: an N-terminal actin-binding domain, an extended rod-shaped domain with four internal 122 amino acid repeats (spectrin-like repeats), and a C-terminal region containing a pair of presumptive helix-loop-helix Ca2+-binding motifs (often referred to as EF-hands ((Blanchard, A., et al., J. Muscle Res. Cell Motil, 10, 280-289 (1989)).
However, the interaction between xcex1-actinin and PKN has not been reported insofar as the present inventors know.
Furthermore, many data recently indicate some signal transduction pathways induced by growth factors overlaps one induced by stress. Rac and Cdc42, other members of the Rho family small GTPases, are activated by not only growth factors but also stresses such as proinflammatory cytokines and ultraviolet radiation, and are involved in the activation of stress activated-MAP kinases (Minden, A. Cell 81, 1147-1157 (1995); Coso, 0. et al., Cell 81, 1137-1146 (1995); and Zhang, S. et al., J. Biol. Chem. 270, 23934-23936 (1995)). Recently, it has been reported that lysophosphatidic acid (LPA), serum, and stresses (for example, arsenite and osmotic shock) regulate c-fos transcription through the activation of serum response element (SRE) by serum response factor (SRF) and that functional Rho is necessary in this case (Hill, C. S. et al., Cell 81. 1159-1170 (1995)). SRE activation, however, is not mediated by other Rho family proteins such as Rac and Cdc42 (Hill, C. S. et al., Cell 81. 1159-1170 (1995)). The above finding suggests the presence of an unknown pathway, responsible for signal transduction to the cell nucleus, downstream of the Rho protein (Vojtek, A. and Cooper, J., Cell 82, 527-529 (1995)).
It should be noted that an asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.
The present inventors have now found that the activated RhoA protein binds to an amino-terminal region of PKN and that the protein kinase activity of PKN is activated in the activated Rho protein-dependent manner. Furthermore, they have found that a particular region of the amino terminal of PKN has an activity to inhibit binding between PKN and the activated Rho protein.
The present inventors have also found that PKN binds to and/or phosphorylates cytoskeletal proteins (intermediate filaments and xcex1-actinin) which control the cell morphology. Specifically, they have found that neurofilament (hereinafter often referred to as NF) L, which is one of the subunits of the neuron-specific intermediate filament, binds to PKN, that the N-terminal regulatory region of PKN binds to head-rod domains of NFL as well as other intermediate filament proteins (other subunits (M and H) of NF and vimentin) or spectrin-like repeats of xcex1-actinin and EF-motif hands, that the purified rat PKN phosphorylates native NF from bovine spinal cord and the head-rod domain of bacterially synthesized intermediate filament proteins (each subunit of NF and vimentin), and that phosphorylation of NFL by PKN inhibits the polymerization of NFL in vitro.
The present inventors have also found that the amino-terminal region of PKN binds to the carboxyl-terminal region of PKN and that a particular region of the amino terminal of PKN acts as a pseudosubstrate for the protein kinase of PKN (i.e., to inhibit the protein kinase activity of PKN).
In addtion, the present inventors have found that PKN is reversibly translocated from cytoplasm into the nucleus by exposing cells to stresses such as heat shock, sodium arsenite and serum starvation.
The present inventions are based on these findings.
Accordingly, an object of the present invention is to provide a peptide comprising a modified amino acid sequence of Protein Kinase N having activated Rho protein binding activity and not having protein kinase activity, and a peptide inhibiting binding between PKN and the Rho protein.
Another object of the present invention is to provide a peptide having cytoskeletal protein (intermediate filament and/or xcex1-actinin) binding activity, a peptide having activity to bind to the protein kinase catalytic region of PKN, a peptide inhibiting the protein kinase activity of PKN, and a peptide inhibiting the translocation of PKN from cytoplasm to nucleus, and a peptide eligible for phosphorylation by PKN.
A further object of the present invention is to provide a base sequence encoding the peptide, a vector comprising the base sequence, a host cell transformed with the vector, a process for producing the peptide or protein, a tumorigenesis or metastasis suppressing agent comprising the protein, and a method for screening a material inhibiting binding between the activated Rho protein and PKN.
In addition, the present inventors have confirmed that the proteins according to the present invention are different from the other Rho protein binding proteins (citron, rhophilin, p160ROCK, Rho-binding kinase, ROKxcex1, rhotekin, myosin-binding subunit, Protein Kinase C1 (PKC1), and 1,3-xcex2-glucan synthase). It should be noted that all the Rho protein binding proteins were identified after the first filed application which provides the right of priority of the present application.